Lupo Maria Giovanna, Bressan Alessandro, Donato Maristella, Canzano Paola, Camera Marina, Poggio Paolo, Greco Maria Francesca, Garofalo Mariangela, De Martin Sara, Panighel Giovanni, Ruscica Massimiliano, Baragetti Andrea, Bollati Valentina, Faggin Elisabetta, Rattazzi Marcello, Catapano Alberico L, Ferri Nicola
Dipartimento di Medicina, Università degli Studi di Padova, Padua, Italy.
Dipartimento di Scienze del Farmaco, Università degli Studi di Padova, Padua, Italy.
Atherosclerosis. 2022 Apr;346:86-97. doi: 10.1016/j.atherosclerosis.2022.01.015. Epub 2022 Jan 22.
A complex interplay among chronic kidney disease (CKD), lipid metabolism and aortic calcification has been recognized. Here we investigated the influence of kidney function on PCSK9 levels and its potential direct action on smooth muscle cells (SMCs) calcification.
In a cohort of 594 subjects, a negative association between glomerular filtration rate and plasma PCSK9 was found. Atherosclerotic cardiovascular disease, as co-morbidity, further increased PCSK9 plasma levels. Diet-induced uremic condition in rats led to aortic calcification and increased total cholesterol and Pcsk9 levels in plasma, livers, and kidneys. Both human and rat SMCs overexpressing human PCSK9 (SMCs), cultured in a pro-calcific environment (2.0 mM or 2.4 mM inorganic phosphate, P) showed a significantly higher extracellular calcium (Ca) deposition compared to control SMCs. The addition of recombinant human PCSK9 did not increase the extracellular calcification of SMCs, suggesting the involvement of intracellular PCSK9. Accordingly, the further challenge with evolocumab did not affect calcium deposition in hSMCs. Under pro-calcific conditions, SMCs released a higher number of extracellular vesicles (EVs) positive for three tetraspanin molecules, such as CD63, CD9, and CD81. EVs derived from SMCs tended to be more enriched in calcium and alkaline phosphatase (ALP), compared to EVs from control SMCs. In addition, PCSK9 has been detected in SMCs-derived EVs. Finally, SMCs showed an increase in pro-calcific markers, namely bone morphogenetic protein 2 and ALP, and a decrease in anti-calcific mediator matrix GLA protein and osteopontin.
Our study reveals a direct role of PCSK9 on vascular calcification induced by higher inorganic phosphate levels associated with renal impairment. This effect appears to be mediated by a switch towards a pro-calcific phenotype of SMCs associated with the release of EVs containing Ca and ALP.
慢性肾脏病（CKD）、脂质代谢和主动脉钙化之间存在复杂的相互作用。在此，我们研究了肾功能对前蛋白转化酶枯草溶菌素9（PCSK9）水平的影响及其对平滑肌细胞（SMC）钙化的潜在直接作用。
在一个594名受试者的队列中，发现肾小球滤过率与血浆PCSK9之间呈负相关。作为合并症的动脉粥样硬化性心血管疾病进一步升高了血浆PCSK9水平。饮食诱导的大鼠尿毒症状态导致主动脉钙化，并使血浆、肝脏和肾脏中的总胆固醇和Pcsk9水平升高。在促钙化环境（2.0 mM或2.4 mM无机磷酸盐，P）中培养的过表达人PCSK9的人及大鼠SMC，与对照SMC相比，细胞外钙（Ca）沉积显著更高。添加重组人PCSK9并未增加SMC的细胞外钙化，提示细胞内PCSK9参与其中。相应地，阿利西尤单抗的进一步刺激并未影响人SMC中的钙沉积。在促钙化条件下，SMC释放出更多对三种四跨膜蛋白分子（如CD63、CD9和CD81）呈阳性的细胞外囊泡（EV）。与对照SMC来源的EV相比，SMC来源的EV往往富含更多的钙和碱性磷酸酶（ALP）。此外，在SMC来源的EV中检测到了PCSK9。最后，SMC中促钙化标志物（即骨形态发生蛋白2和ALP）增加，而抗钙化介质基质Gla蛋白和骨桥蛋白减少。
我们的研究揭示了PCSK9在与肾功能损害相关的较高无机磷酸盐水平诱导的血管钙化中起直接作用。这种作用似乎是由SMC向促钙化表型的转变介导的，该转变与含有Ca和ALP的EV释放有关。
